Liquid chromatograph mass spectrometer

ABSTRACT

In a liquid chromatograph mass spectrometer, chromatogram data is obtained by carrying out alternately a mass scanning in a positive ion detection mode and a mass scanning in a negative ion detection mode, obtaining the chromatogram data in every positive and negative polarities through summing up the mass spectrum data obtained at the respective mass scannings, and adding the data of both polarities. Since the chromatograms in both polarities are averaged, even if there is a level difference therebetween, a chromatogram in a normal form can be obtained.

BACKGROUND OF THE INVENTION AND RELATED ART STATEMENT

[0001] The invention relates to a liquid chromatograph massspectrometer, in particular, an. apparatus suitable for fractionatingvarious components contained in a sample solution by using a liquidchromatograph mass spectrometer.

[0002] Heretofore, there has been known a fraction chromatograph whereina plurality of components contained in a sample is separated andcollected by using a chromatograph device, such as high performanceliquid chromatograph (hereinafter referred to as “HPLC”).

[0003]FIG. 5 is a block diagram for showing an example of a structure ofa fraction chromatograph using HPLC. An eluant, i.e. mobile phase,stored in an eluant tank 1 is sucked by a pump 2 and is transferred toflow into a column 4 through a sample introduction portion 3 at apredetermined flow rate. A sample solution injected into the mobilephase at the sample introduction portion 3 is introduced into the column4 together with the mobile phase, and while passing through the column4, its components are separated and eluted. A detector 5 detectssequentially the components eluted from the column 4 and sends detectionsignals to a signal process portion 6. All or a part of the eluatepassing through the detector 5 is introduced into a fraction collector8. The signal process portion 6 prepares a chromatogram based on thedetection signals obtained from the detector 5, and a control portion 7provides the fraction collector 8 with a control signal for fractionbased on a peak appearing on the chromatogram at real time. The fractioncollector 8 controls an electromagnetic valve and the like based on thecontrol signal and distributes the eluates to vials corresponding to therespective components.

[0004] By the way, recently, there has been widely used a liquidchromatograph mass spectrometer (hereinafter referred to as “LC/MS”)using a mass spectrometer (hereinafter referred to as “MS”) as adetector of HPLC. In MS, since various components contained in anintroduced sample are separated and detected in every mass number, i.e.mass/charge, even if a plurality of components is timewise overlapped,there is an advantage such that these components are separated andsubjected to a qualitative analysis and a quantitative analysis.

[0005] In the LC/MS, a mass spectrum can be obtained by carrying out amass scanning over a set mass region, sequentially detecting strengthsof ions separated in every mass number and examining a relationshipbetween the mass number and the strength. Also, a total ion chromatogram(hereinafter referred to simply as “chromatogram”) can be obtained byrepeatedly carrying out the mass scannings, integrating the ion strengthin every scanning regardless of the mass number and examining a timewisechange of the total ion strength. Further, by watching a specific massnumber, a mass chromatogram can be obtained by examining a timewisechange of the strengths of ions having the mass number in everyscanning.

[0006] In case the LC/MS, as described above, is used for the fractionchromatograph, it is necessary to determine a timing of fraction basedon the chromatogram data for preparing a chromatogram or masschromatogram. Normally, the chromatogram data is calculated according toprocess conditions set beforehand (for example, strengths of ions havinga specific mass number are added, strengths of ions in a predeterminedmass region are added and the like), from a great number of massspectrum data obtained by one-time mass scanning. Thus, a chromatogramdata can be obtained in every mass scanning. Therefore, for example, incase a spectrometry is carried out while alternatively changing a massscanning in a positive ion detection mode for detecting positive ionsand a mass scanning in a negative ion detection mode for detectingnegative ions, a chromatogram data obtained at a certain time point t isa value calculated based on the mass spectrum obtained by the positiveion detection mode, and the subsequent chromatogram datum obtained att+Δt is a value calculated based on the mass spectrum obtained by thenegative ion detection mode.

[0007] Generally, since the levels of the background noises in thepositive ion detection mode and the negative ion detection mode aredifferent, the chromatogram prepared based on the mass spectrum obtainedin the positive ion detection mode and the chromatogram prepared basedon the mass spectrum obtained in the negative ion detection mode aredifferent in levels of the base lines as shown in FIG. 6(a). Therefore,when the chromatogram data obtained when the positive polarity and thenegative polarity are switched over as described above is connected oradded in time sequence, the chromatogram curve having sawteeth shapes asshown in FIG. 6(b) is obtained. Also, in case a component detectableonly by the positive ion detection mode and a component detectable onlyby the negative ion detection mode are mixed, since the respectivechromatograms become, for example, as shown in FIG. 7(a), when thechromatogram data obtained when the positive polarity and the negativepolarity are switched over are connected or added in time sequence, thepeak waveform becomes sawteeth shapes as shown in FIG. 7(b).

[0008] In either case, in the control portion 7, since an accuratestarting point and an accurate terminal point of the peak can not bedetermined by using such chromatogram data, it is impossible todetermine the timing of fractionating the respective components, or anerroneous control signal is sent to the fraction collector 8. In view ofthe defects as described above, in case the fraction operation iscarried out by the conventional LC/MS, since the fraction operation cannot be carried out while alternately changing the positive polarity andthe negative polarity, it is necessary that the fraction operation inthe positive ion detection mode and the fraction operation in thenegative ion detection mode are separately carried out. Thus, thefraction operation is not carried out effectively.

[0009] In view of the above problems, the present invention has beenmade and an object of the present invention is to provide a liquidchromatograph mass spectrometer, wherein even in case a massspectrometry is carried out while changing the spectrometry conditions,such as alternately changing a positive polarity and a negativepolarity, a chromatogram for normally operating a fraction collector canbe obtained, so that a proper fraction operation can be done by onlyone-time spectrometry.

[0010] Further objects and advantages of the invention will be apparentfrom the following description of the invention.

SUMMARY OF THE INVENTION

[0011] In order to solve the above-stated problems, according to thepresent invention, in a liquid chromatograph mass spectrometer, asample, components of which are separated in a liquid chromatographportion in a time-wise direction, i.e. along a passage of time, isintroduced into a mass spectrometry portion and a fraction collector,and the fraction collector fractionates and collects the respectivecomponents based on the information obtained in the mass spectrometryportion. The liquid chromatograph mass spectrometer includes: a settingdevice for setting beforehand a plurality of spectrometry conditionswhen a mass spectrometry is carried out; a spectrometry execution devicefor executing a cycle of spectrometry by changing the spectrometrycondition set by the setting device whenever one-time mass scanning inone cycle is carried out, the periodical spectrometry being repeatedsequentially; an operation device for obtaining chromatogram data byadding together a number of mass spectrum data obtained by the one-timemass scanning whenever the cycle of spectrometry is completed andfurther adding thereto values in the respective mass scannings, or forobtaining the chromatogram data by adding the mass spectrum data withrespect to a specific mass number obtained by the respective massscannings; and a fraction control device for controlling an operation ofthe fraction collector based on the chromatogram data obtained by theoperation device.

[0012] Here, the “spectrometry condition” means a condition which has aneffect on the ion generating condition or the ion detecting condition.For example, the spectrometry condition may include a positive iondetection mode for detecting a positive ion and a negative ion detectionmode for detecting a negative ion. When the positive ion detection modeand the negative ion detection mode are set by the setting device, thespectrometry execution device carries out alternately one-time massscanning of the positive ion detection mode and one-time mass scanningof the negative ion detection mode. Since a large number of massspectrum data with respect to the mass number in a predetermined regioncan be obtained by the respective mass scannings, the operation deviceadds the mass spectrum data in each polarity, and further adds theretothe values of the respective polarities to obtain a single chromatogram.More specifically, since the single chromatogram reflects a plurality ofmass spectrum data of both polarities, even if the levels on the baselines of the respective chromatograms of the positive and negative ionsare different, or a peak is present only in either the positive ion ornegative ion chromatogram, they are averaged.

[0013] Incidentally, in case a fraction operation is carried out basedon the mass chromatogram, the operation device adds the mass spectrumdata with respect to a specific mass number obtained by each massscanning to obtain the chromatogram data.

[0014] According to the liquid chromatograph mass spectrometer of thepresent invention, even in case the mass scanning is carried out whilechanging the spectrometry conditions of the mass spectrometry, there canbe obtained a chromatogram wherein the peak waveform can be normallyobtained, so that the fractions of the respective components can beproperly carried out by the fraction collector. Also, for example, sincethe fraction operations with respect to the components of bothpolarities can be carried out by a single spectrometry, it is notnecessary to carry out fraction operations for the respective polaritiesas in the conventional liquid chromatograph mass spectrometer, thusshortening the time required for the fraction operation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a block diagram for showing an entire structure of LC/MSof an embodiment according to the present invention;

[0016]FIG. 2 is a graphic chart for explaining a signal processoperation of the present embodiment;

[0017]FIG. 3 is a flow chart for showing the signal process operation ofthe present embodiment;

[0018] FIGS. 4(a) and 4(b) are examples of chromatograms obtained in thepresent embodiment;

[0019]FIG. 5 is a block diagram for showing a structure of a fractionchromatograph using a general HPLC;

[0020] FIGS. 6(a) and 6(b) are chromatograms for explaining problems ina fraction device using a conventional LC/MS; and

[0021] FIGS. 7(a), 7(b) are other chromatograms for explaining problemsin a fraction device using a conventional LC/MS.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Hereunder, LC/MS of an embodiment of the present invention willbe explained with reference to FIG. 1 to FIG. 4(b).

[0023]FIG. 1 is a block diagram of an entire LC/MS according to thepresent embodiment. A sample liquid eluted from a column 4 of an LC isdivided into two-flow paths at a predetermined ratio at a flow-pathdiverging portion 9, one of which is sent to an MS portion 10 and theother of which is sent to a fraction collector 8. The MS portion 10includes a nebulizing or atomizing chamber 11 having a nozzle 12 and adischarge electrode 13, and a spectrometry chamber 16 having aquadrupole filter 17 and an ion detector 18. There are provided twointermediate chambers 15 between the nebulizing chamber 11 and thespectrometry chamber 16. The nebulizing chamber 11 and the firstintermediate chamber 15 are connected through a desolvent pipe 14. Thesignal detected by the ion detector 18 in the MS portion 10 is inputtedinto a signal process portion 20, and, after being subjected toprocessing as described later at the signal process portion 20, givesthe chromatogram data to a control portion 21. The control portion 21controls operations of the respective portions in the MS portion 10, thefraction collector 8, and operations of the respective portions of theLC though control signal lines are not shown,.

[0024] Operations of the MS portion 10 are as follows. When the samplesolution supplied from the column 4 reaches the nozzle 12, the samplesolution is atomized in the nebulizing chamber 11 as high temperaturedrops. The dispersed drops collide with gas molecules under theatmospheric pressure, are smashed into further fine drops, and quicklydried, i.e. removal of the solvent, to thereby vaporize the samplemolecules. The fine gas particles contact the buffer ions produced bythe corona discharge from the discharge electrode 13 to cause a chemicalreaction, and ionized. The fine drops containing the generated ionsplunge into the desolvent pipe 14 and are further subjected to thedesolvent while the fine drops pass through the desolvent pipe 14. Theions are sent to the spectrometry chamber 16 through the twointermediate chambers 15, and only objective ions having a specific massnumber, i.e. mass/charge, pass through the quadrupole filter 17 disposedin the spectrometry chamber 16 to reach the ion detector 18. Electriccurrent corresponding to the ion number which has arrived at the iondetector 18 can be taken out therefrom.

[0025] In the MS portion 10, a positive ion detection mode for detectingthe positive ions by generating the positive ions and a negative iondetection mode for detecting the negative ions by generating thenegative ions can be switched over in a short time, by changing voltagesapplied to the respective portions, such as the discharge electrode 13,and switching the operation of the ion detector 18.

[0026] Hereunder, operations of the present LC/MS when fractionoperations are carried out in both positive and negative polaritiesalternately will be explained.

[0027]FIG. 3 is a flow chart for showing operations at the time of thespectrometry in the signal process portion 20 and the control portion21, and FIG. 2 is a graphic chart for explaining the operations thereof.An operator inputs various parameters, such as operation conditions ofLC, operation conditions of the MS portion 10 and process conditions inthe signal process portion 20, to set therein from the operating portion22. These conditions include a mass region at a time of mass scanning, amass step, a scanning time and so on in the MS portion 10.

[0028] When the spectrometry starts, first, the control portion 21 setsparameters of the respective portions of the MS portion 10 to be thepositive ion detection mode (Step S1), and carries out the mass scanningin a predetermined mass region (Step S2). At the time of the massscanning, when the voltage applied to the quadrupole filter 17 iscontrolled, the mass number of the ions having passed through thequadrupole filter 17 and arrived at the ion detector 18 is changed. Thesignal process portion 20 processes the detection signals which aresequentially changed at the time of the mass scanning, and obtains themass spectrum data for showing relationships between the mass number andthe ion strength (Step S3). The mass spectrum reflects only the positiveion strength as shown in FIG. 2. Among a large number of mass spectrumdata, the mass spectrum data is extracted according to the predeterminedprocess conditions, such as mass region, and is added together to obtainchromatogram data A(+) of the positive polarity and stored in a memory(Step S4).

[0029] Then, the control portion 21 sets parameters of the respectiveportions of the MS portion 10 to become the negative ion detection mode(Step S5), and carries out the mass scanning in a predetermined massregion (Step S6). More specifically, the control portion 21 carries outthe mass scanning in the same manner as in the above-explained positiveion detection mode, and the signal process portion 20 processes thedetection signals which are sequentially changed at the time of massscanning to obtain the mass spectrum data showing relationships betweenthe mass number and the ion strength (Step S7). The mass spectrumreflects only the negative ion strength as shown in FIG. 2. Among alarge number of mass spectrum data, the mass spectrum data is extractedaccording to the predetermined process conditions, and is added togetherto obtain chromatogram data A(−) of the negative polarity (Step S8).Then, when the chromatogram data A(+) and A(−) of the positive andnegative polarities are completed, both data is added together to obtainthe chromatogram data A and outputted as an analogue value (Step S9).Thereafter, until spectrometries of all components are completed, theabove-described processes are repeated by returning from Step S10 toStep S.

[0030] With the above-described process, as shown in FIG. 2, achromatogram datum A can be obtained in every two times of the massscannings (one for the positive polarity and one for the negativepolarity). FIGS. 4(a) and 4(b) are chromatograms prepared based on thechromatogram data obtained from the signal process portion 20. Accordingto the present LC/MS, even in case the chromatograms of the positive andnegative polarities are separately prepared and the respective peaks areformed in different positions as shown in FIG. 4(a), the peaks of bothpolarities appear on the chromatogram in a normal form as shown in FIG.4(b), and the sawteeth shapes as shown in FIG. 7(b) are not formed.

[0031] When the control portion 21 receives the chromatogram data fromthe signal process portion 20 at a real time, the control portion 21detects a starting point of a peak of an objective component to befractionated and outputs a collection start signal to the fractioncollector 8 with a predetermined time delay from the time when thestarting point is detected. The time delay is determined by a flow rateof a mobile phase and pipe capacities from the flow path divergingportion 9 to the nozzle 12 of the MS portion 10 and from the flow pathdiverging portion 9 to an electromagnetic valve of the fractioncollector 8. In the fraction collector 8, when the objective componentarrives at the electromagnetic valve, the electromagnetic valve isopened according to the collecting start signal to start fraction. Whena termination point of the peak of the objective component is detected,the control portion 21 sends a collection completion signal to thefraction collector 8 in the same manner. Thus, when the fraction orseparation of the objective component is completed, the electromagneticvalve is closed. In case a plurality of components is fractionated,during a period when the electromagnetic valve is closed, a vial bottleis moved by a biaxial arm or the like and an empty vial bottle is set ata fractioning position for the next fraction.

[0032] Incidentally, in case a spectrometry is carried out by using onlyone polarity without changing the positive polarity and the negativepolarity as described above, either the chromatogram datum A(+) orchromatogram datum A(−) in FIG. 3 may be processed as zero.

[0033] While the above embodiment shows the case where the positivepolarity and the negative polarity are changed, in addition to this, thesame method can be used by changing or shifting the operation conditionsof the various mass spectrometries. For example, it is possible to carryout the respective mass spectrometries through change of a mode forcleavage of ions by changing a voltage to be applied to a deflectorelectrode disposed in the intermediate chamber of the MS portion 10. Ofcourse, in case the operation conditions include more than three kinds,chromatogram data may be calculated in every mass spectrometries of morethan three times corresponding to the operation conditions.

[0034] While the invention has been explained with reference to thespecific embodiments of the invention, the explanation is illustrativeand the invention is limited only by the appended claims.

What is claimed is:
 1. A chromatograph mass spectrometer forsequentially processing a sample, comprising: a setting device forsetting beforehand a plurality of spectrometry conditions when a massspectrometry is carried out, a spectrometry execution deviceelectrically connected to the setting device for executing a cycle ofspectrometries by changing one of the spectrometry conditions set by thesetting device whenever one mass scanning in said cycle is carried out,said spectrometry execution device sequentially executing the one cyclespectrometries repeatedly, and an operation device electricallyconnected to the spectrometry execution device for obtainingchromatogram data by respectively adding together a number of massspectrum data obtained by the one mass scanning in one cycle when onecycle of the mass spectrometry is completed.
 2. A chromatograph massspectrometer according to claim 1, further comprising an introducingsection for introducing the sample, a chromatograph portion connected tothe introducing section f or separating components in the sample along apassage of time, a mass spectrometry portion connected to thechromatograph portion f or analyzing the components, and a fractioncollector connected to the chromatograph portion for collecting therespective components based on information obtained in the massspectrometry portion.
 3. A chromatograph mass spectrometer according toclaim 2, further comprising a fraction control device electricallyconnected to the operation device for controlling an operation of thefraction collector based on the chromatogram data obtained by theoperation device.
 4. A chromatograph mass spectrometer according toclaim 3, wherein said operation device adds the chromatogram data at theone mass scanning with an added value in another mass scanning.
 5. Achromatograph mass spectrometer according to claim 3, wherein saidoperating device obtains the chromatogram data by adding the massspectrum data with respect to a specific mass number obtained by eachmass scanning.
 6. A chromatograph mass spectrometer according to claim1, wherein said mass spectrometer is a liquid chromatograph massspectrometer.